1. Institute of Laboratory Animal Science University of Zurich Transgenic Mouse Models Bio 426 / HS 2012 Kurt Bürki, Pawel Pelczar Institute of Laboratory Animal Science, University of Zurich

2. Institute of Laboratory Animal Science University of Zurich Goals To cover the techniques to generate transgenic models To compare advantages / disadvantages / limits of the techniques To discuss important models in several fields of biomedical research

3. Institute of Laboratory Animal Science University of Zurich Learning Objectives By the end of the lecture series the participants are able to: List advantages and disadvantages of the major methods to generate transgenic animals Design functional transgenes and targeting vectors Present and critically discuss original papers in the field in a comprehensive form (key skill)

4. Institute of Laboratory Animal Science University of Zurich Additional Practical Courses BIO 413: Generation of Transgenic Animals (LTK Module 3E) BIO 412: Einführung in die Labortierkunde / Introduction into Laboratory Animal Science (LTK Modul 1)

5. Institute of Laboratory Animal Science University of Zurich Structure of the Lecture Series Introduction Technical Aspects (Students: Paper to read / Comrehensive presentation) Transgenic Mouse Models (Students: Paper or review to read / Identification of questions relevant for a given field) Exam Visit of a Laboratory

6. Institute of Laboratory Animal Science University of Zurich Transgenic Animals: Definition Mutant animals carrying experimentally introduced foreign genetic elements in all their cells, including the germline

7. Institute of Laboratory Animal Science University of Zurich Steps towards a Transgenic Model Working hypothesis Gene Construct Insertion into an early embryonic stage Screening for transgenic animals Profiling of expression pattern Phenotyping Model Validation / Experimentation

8. Institute of Laboratory Animal Science University of Zurich Gene Construct Expression constructs (transgenes) Viral vectors: retroviral/lentiviral vectors Targeting constructs: comprising homologies to murine sequences

9. Institute of Laboratory Animal Science University of Zurich Gene Insertion Insertion by nuclear DNA repair / recombination mechanisms Random (non-homologous end joining NHEJ: subject to position effects) Targeted (homologous recombination)

10. The Mouse genome

11. Institute of Laboratory Animal Science University of Zurich Genetic Networks GenesPhenotype

12. Institute of Laboratory Animal Science University of Zurich

14. Transgenics vs. Genetics Transgene Promoter/Coding Sequence Insertion Site Targeting Vector Knock-out/Knock-in Conditional Mutants Phenotype Loci, Genes Position Effects Variegated Expression Penetrance Expressivity Polygenic Traits Genetic Background Phenotype

15. Institute of Laboratory Animal Science University of Zurich Genome Mammalian 20 chromosomes 2.6 Gb ~25000 genes 99% have human counterpart Strains Inbred Outbred Recombinant inbred Consomic Fluorescent Life Cycle 4-day oestrus 20-day gestation 4-8 pups per litter 2-8 litters per female 7 weeks to sexual maturity 2-3 year lifespan Reverse genetics Knockouts Transfenics Conditional expression Inducible expression Retroviral vectors siRNA The Mouse as an Experimental System Assisted reproduction Cryopreservation Embryo rederivation In vitro fertilization Intracytoplasmic sperm injection Cloning Tools Genome sequence Embryonic stem cells Expression arrays Gene-trap libraries Insertional vector libraries BAC libraries

16. Institute of Laboratory Animal Science University of Zurich Why the Mouse? The closest to humans – mammal The most complex - integration of systems (endocrine, immune, nervous etc.) Of the model organisms which may be genetically modified, the mouse is: Genetic manipulation is extremely versatile – Gain-of-Function (Transgenesis), Loss-of-Function (knock-out), Change-of-Function (knock-in); temporally and spatially restricted (conditional)

17. Institute of Laboratory Animal Science University of Zurich Applications of transgenic mice Transgenic mice are often generated to address the role a gene plays in a biological process at the level of the whole organism: - To confirm the role of a gene mutation - To help unravel the molecular mechanisms that control gene expression - To help unravel the biochemical in vivo mechanisms and the origin of disease - To develop an animal model to test therapeutic strategies

18. Institute of Laboratory Animal Science University of Zurich Transgenic Animals: Methods Classical - Pronuclear Microinjection - Lentiviral Infection - Embryonic Stem (ES) Cell Gene Transfer - ES Cell mediated Gene Targeting (knock-out, knock-in) Experimental - Transfection of Somatic Cells - Cloning - Sperm Based Transfection (ICSI) - Transposons

19. Institute of Laboratory Animal Science University of Zurich Generation of transgenic animals

20. Institute of Laboratory Animal Science University of Zurich Mouse Transgenesis Methods Pronuclear microinjection Lentivral infection ES based transgenesis pros Relatively simple and efficient Long transgenes possible Potentially all species Very efficient Single copy insertions No technical equipment Works in many species Long transgenes possible Gene targeting possible Single copy insertions cons Random integration Multicopy insertions ( Strain limitations) High embryo mortality 9.5 kb packaging limit Safety issues (?) Only random integration Technically difficult Time consuming Species / Strain limitations

21. Institute of Laboratory Animal Science University of Zurich Pronuclear Microinjection Microinjection of DNA directly into the pronuclei of fertilized eggs Implantation of the microinjected eggs into a surrogate mother Allowing the embryos to develop to birth Demonstrating that the foreign gene has been stably incorporated into the host genome and that it is heritable in at least one of the offspring Demonstrating that the gene is expressed and regulated correctly in the host organism

22. Institute of Laboratory Animal Science University of Zurich Microinjection Station

23. Institute of Laboratory Animal Science University of Zurich Blastocysts d 3-4ES-Cell-Colonies Establishment of ES Cells in vitro ICM (Innere Zellmasse)

24. Institute of Laboratory Animal Science University of Zurich Germline male chimera (C57BL/6 in BALB/c) with offspring

25. Institute of Laboratory Animal Science University of Zurich Mate founders 024810126 DNA or LV injection gestation maturation of founders Timeline: Transgenesis by Pronuclear Microinjection or Lentiviral transfection gestation Birth Identyfy founders maturation of F1 progeny Begin analysis

26. Institute of Laboratory Animal Science University of Zurich Timeline: generation of ES cell-derived mice Introduce targeting vector into ES cells Identify homologous recombinants by DNA analysis Identify mouse Chimeras with high ES cell contribution Germline transmission Begin analysis 024810126 Drug selection Colony growth and expansion Inject clones into blastocysts Sexual maturation of chimeras Identify male and female heterozygotes Sexual maturation of heterozygotes Identify homozygotes gestation

27. Institute of Laboratory Animal Science University of Zurich Trends in the Field of Transgenic Animals More Refined Transgene Systems: - temporal regulation (tet ON/OFF) - tissue specific and temporal regulation ( Cre/lox) Gene Targeting in Species other than the Mouse Integrative Databases Animal Welfare Aspects

28. Institute of Laboratory Animal Science University of Zurich Trends with Transgenic Animals (1) Targeted Modifications, Control over Expression or Silencing, Combined (binary) Systems Inducible Transgene-Expression Tet-on, Tet-off Systems) Tissue-specific knock-outs (Cre-lox System) Inducible knock-outs (CreMER System)

29. Institute of Laboratory Animal Science University of Zurich Trends with Transgenic Animals (2) Routine Gene-Targeting in Mammalian Species other than the Mouse New: Gene Targeting in Rat ES Cells / iPS Cells / Spermatogonial Stem Cells New: Zinc-Finger Nucleases for the Introduction of Site- Directed Genome Modifications

30. Institute of Laboratory Animal Science University of Zurich Transgenic Animals: Potential Problems Technical problems to closely mimic a desired situation Underestimation of biological complexity Mouse – Human differences Inappropriate analysis Undefined genetic backgrounds

31. Institute of Laboratory Animal Science University of Zurich Example: The App Gene (Alzheimers Disease)

32. Institute of Laboratory Animal Science University of Zurich Paper to Read Brinster R.L. et al.: Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci USA 82, 4438-4442 (1985).